Fig 1.
The main steps of the procedure include maintenance of bean and spider mite populations (Step 1), collection of the adult female mites (Step 2), preparation of T. urticae larvae and adult female mites tightly synchronized in their development (Step 3) and methods for delivery of small molecules (Step 4). The respective figures, tables, supplementary figures and supplementary videos are referenced.
Fig 2.
Preparation of mites synchronized in development.
(A) Synchronization of larval emergence. Schematic overview of the protocol for the synchronous hatching of spider mite eggs. (B) Synchronization of adult emergence. Schematic overview of the protocol for the synchronous molting of spider mite deutonymphs, which is the last nymph stage in mite development.
Fig 3.
Synchronization of mite development.
(A) Synchronization of egg hatching. Relative egg hatchability in: Control, 4-day-old eggs in 50% RH (n = 120–271 with 3 replicates); Submerged, eggs submerged in water for 4 days (n = 86–142 with 3 replicates); Post water removal, eggs submerged in water for 4 days, then drained and incubated for 3 and 24 h in 50% RH (n = 85–137 with 3 replicates). All samples were incubated at 26°C. Data are shown as mean ± SE. Values with different letters differ significantly in the cumulative hatchability (p<0.05, Tukey’s HSD test after one-way ANOVA). (B) Relative adult emergence from deutonymphs under different environments (n = 10 with 4 replicates). Day 1 and Day 2, adult emergence from deutonymphs kept at 18°C in cups that were covered with vented (Control) and non-vented (No ventilation) lids after 1 and 2 days respectively. Day 2 (open lid, 30 min), adult emergence from deutonymphs kept at 18°C in cups that were covered with vented (Control) and non-vented (No ventilation) lids for 2 days and transferred to 26°C and 50% relative humidity environment for 30 min. Data are shown as mean ± SE. Data at each time point were compared using the Wilcoxon–Mann–Whitney test (no asterisk, p > 0.05; *, p < 0.05).
Fig 4.
Spider mite larvae and adult females feeding for 24 h on an artificial diet delivered through Parafilm® M hemispheres.
(A) Diet supplemented with the 3% blue food dye. (B) Diet supplemented with 100 ng/μL Alexa Fluor 488 green-fluorescent dye. Mite images in (A) represent the control on the left and mites feeding in the presence of the 3% blue food dye on the right. Images in (B) were taken with an exposure time of 0.4 sec (ISO: 400). Differential interference contrast (DIC) and fluorescent images were merged at 50% opacity with ImageJ in (B) [32].
Fig 5.
Analysis of the mite survivorship during a 5-day feeding period on artificial diet.
(A) Survival of newly-hatched larvae (n = 113). (B) Survival of adult females (n = 57). Survival curves were plotted with the Kaplan–Meier method.
Table 1.
Effect of Silwet L-77 on solution properties and mite behavior at 24 and 48 h after feeding on leaf discs coated with water or Silwet L-77 (0.0125, 0.025 and 0.05%, v/v) at 26°C.
Fig 6.
Accumulation of tracer dyes in adult female mites after feeding for 48 h on a leaf disc.
Leaf disc was coated with: (A) water (left, Control) and 3% blue food dye (right, Treatment); or, (B) fluorescent Alexa Fluor 488 dye (100 ng/μL). Images in (B) were taken with an exposure time of 0.4 sec (ISO: 400). Differential interference contrast (DIC) and fluorescent images were merged at 50% opacity with ImageJ [32].
Fig 7.
Analysis of mite performance while feeding on bean leaf discs coated with water or Silwet L-77.
Larval (A) and adult female (B) survivorship during a 6-day feeding on bean leaf discs coated with water or Silwet L-77. Sample: (A) water control (solid line, n = 125; 0.025% Silwet L-77 (dashed line, n = 140); (B) water control (black line, n = 27); 0.025% Silwet L-77 (dashed line, n = 32). Survival curves were plotted with the Kaplan–Meier method and compared with the log-rank test. (C) Fecundity of females during a 6-days feeding period on bean leaf discs coated with water (n = 10) or 0.025% Silwet L-77 (n = 15). Results are shown as mean ± SE. Difference between treatments was tested with t-test, P = 1. Coated leaf discs were replaced every other day (A-C).
Table 2.
Survival of adult T. urticae females at 24 and 72 h after recovery from soaking in 0.1% Tween 20 or 0.1% Triton X-100 (v/v) at 20°C for 24 h.
Table 3.
Survival of T. urticae larvae at 24 and 72 h after recovery from soaking in 0.1% Tween 20 or 0.1% Triton X-100 (v/v) at 20°C for 0, 2, 4, or 24 h.
Fig 8.
Accumulation of tracer dyes in mites after recovery from soaking.
(A) for 24 h at 20°C, in 0.1% Triton X-100 (Control) and blue food dye (0.1% Triton X-100 + 3% blue food dye); (B) for 24 h at 20°C, in 0.1% Triton X-100 (Control) and fluorescent dye (0.1% Triton X-100 + 100 ng/μL Alexa Fluor 488); (C) for 24 h at 4°C, in 0.1% Triton X-100 (Control) and fluorescent dye (0.1% Triton X-100 + 100 ng/μL Alexa Fluor 488); and (D) for 10 minutes at 4°C, in 0.1% Triton X-100 (Control) and fluorescent dye (0.1% Triton X-100 + 100 ng/μL Alexa Fluor 488). The fluorescent images (in B-D) were taken with and an exposure time of 0.4 s (ISO: 400). The differential interference contrast (DIC) and fluorescent images were merged at opacity of 50% with ImageJ [32].